The final step in progression from the rational design to assessment of proper-ties of ARC-type inhibitors was confirmation of the ability of the latters to implement their high affinity and inhibition potential towards target PKs in bio-logically complex systems such as tissues and organs. As an example system, the smooth muscle tissue of rat cerebellum isolated intact arteries was chosen, due to the high content of PKAc and PKGI, and a wealth of information in literature about effects resulting from inhibition of these PKs in this tissue.
First of all, the ability of ARC-Photos (ARC-583, 669, and ARC-1059) to penetrate plasma membrane of smooth muscle cells in isolated arteries was tested. In accordance with the previously reported experiments in HEK-293 and CHO cell cultures [Uri et al., 2002; Vaasa et al., 2010], all compounds demonstrated cellular uptake after 1 h incubation of arteries in 10 M solution of ARCs at room temperature (Figure 50). The staining was characteristically pronounced in some regions of the arteries, and both, nuclear staining and diffused distribution in the cytoplasm could be indicated, whereas the pro-portion of distribution between the cytoplasm and the nucleus was dependent on the structure of the tested ARC (i.e., ARC-669 accumulated to nucleus, but ARC-583 localization was more diffused, although still demonstrating nuclear inclusion). The studies of time- and concentration-dependent uptake of ARC-1059 revealed that at a concentration of 10 M, the internalization of ARC was already observable after 20 min of incubation, although the nucleus was not yet stained (please refer to Figure 6 from Paper 4). By contrast, at 1 M or lower concentrations of ARC-1059, only minor extent of internalization was observed even after 60 min of incubation. The latter observation is also in good accor-dance with experiments in cell cultures [Vaasa et al., 2010], which also indicated that a “critical” threshold of the ARC-Photo concentration had to be crossed in order to obtain effective internalization of conjugates into the cells.
Figure 50. Staining of isolated rat cerebellum arteries by (A) 10 M ARC-583, (B) 10 M ARC-669, or (C) 10 M ARC-1059 (in all cases, incubation time one hour at room temperature).
A B C
Next, measurement of the ability of ARC-903 to inhibit PKGI and PKAc at physiological conditions was performed by a pressure myography assay in the arteries isolated from rat cerebellum. Both PKGI and PKAc trigger vasodilation via several mechanisms, with the net outcome in all cases represented by dephosphorylation of MLC20; the inhibition of PKGI or PKAc should therefore induce vasoconstriction (Scheme 1, Scheme 2 from the Section 1.2.2. PKG).
Previously, vasoconstrictive effects had been demonstrated for the PKGI-selective potent PCI D-DT-2 (concentration of 1...2 M in the superfusate; [Nickl et al., 2010]), which relieved PKG- but not PKAc-induced vasodilation. The experimental set had comprised an isolated artery cannulised on glass pipettes and developing sustained myogenic tone upon pressurization to 80 mmHg. The change of the arterial diameter had been monitored in response to selective activators of PKG or PKAc (in order to discriminate the components of vasodilation triggered by either PK) and inhibitors applied via the superfusate. This time, the same technique was applied in tests with 903 (concentration of 4 M in the superfusate). ARC-903 successfully abolished vasodilation induced by activators of both, PKGI (i.e., 8-Br-cGMP, MAHMA-NONOate) and PKAc (i.e., Forskolin, 8-CPT-cAMP), confirming the ability of potent ARCs to inhibit PKs in physiological conditions (please refer to Figure 7 from Paper 4). Furthermore, the vasoconstriction of arteries induced by ARC-903 occurred to an extent below the initial (basal) diameter, indicating that the basal activity of PKs was also inhibited; the latter result could also be repeated when ARC-903 was applied to non-dilated artery.
Prior to the pressure myography experiments, ARC-903 had revealed in bioanalytical in vitro assays highly potent inhibition of not only PKAc and PKG, but also ROCK [Enkvist et al., 2006]. The promiscuous nature of ARC-903 was therefore considered problematic, as in smooth muscle tissue, the pathways of cyclic-nucleotide dependent PKs oppose pathways of ROCK; it was therefore relieving to witness that ARC-903 could still implement its high potency via inhibiting PKAc and PKGI pathways in tissue. The reason why effects of inhibition of PKA and PKGI on the one hand, and ROCK on the other hand did not mutually even out, probably lies in the fact that PKAc and PKG serve themselves as deactivators of ROCK, and therefore the latter was “switched off”
during the whole course the experiment. In turn, the latter fact means that the constriction of artery upon application of ARC-903 was achieved via ROCK-independent mechanisms, which might involve downregulation of telokin-induced MYPT activity and re-gain of function of Ca2+-pathways.
The application of a group-selective inhibitor with high affinity towards PKAc and PKGI but not ROCK would probably yield more drastic and sustained effects in the given assay; still, this final set of pressure myography experiments convincingly demonstrated the wide field of application possi-bilities for the promiscuous nature of ARC-conjugates. Moreover, the ability of ARC-903 to inhibit PKGI- and PKAc-induced vasodilation in the arteries isolated from rat cerebellum represented the first clearly pronounced physiolo-gical effect of BSIs reported so far.
CONCLUSIONS
The present thesis focussed on design, synthesis and in vitro characterization of new conjugates of an adenosine analogue and an arginine-rich peptide as inhi-bitors of basophilic protein kinases. The progress within the given work comes to the fore by step-by-step evolution of ARCs from submicromolar to sub-nanomolar inhibitors of PKAc, PKG, ROCK and several other representatives of the AGC-group. Importantly, by virtue of enhanced proteolytic stability and relatively good cell membrane-penetrative properties, the most efficient ARCs are applicable both in biochemical assays and in living cells or tissues. Depen-dent on the character of application, different ARCs may be used as generic probes of basophilic PKs or as remarkably selective modulators of PK activity.
The main results of the thesis can be summarized as follows:
Two new sets of ARCs (subsequently termed as ARC-II and ARC-III) have been designed and synthesized. The main modifications in the structure of new compounds as compared to the set of the initial ARCs (ARC-I) were as follows:
o In case of ARC-II, the C-terminus of the compounds was amidated, D-Arg residues in the peptidic fragment were substituted for L-Arg residues, and H-9 moiety was incorporated as nucleosidic fragment.
The most efficient representative of ARC-II was ARC-903 with inhibition IC50 value of 5.3 nM towards PKAc.
o In case of ARC-III, a second linker and the chiral moiety (amino acid) were introduced. The most efficient representative of ARC-III was ARC-1028 with competitive displacement IC50 value of less than 0.5 nM towards PKAc.
The bisubstrate character of ARCs was confirmed in displacement stu-dies with PKAc (where the fluorescent ARC-probe could be success-fully displaced from its complex with the kinase by compounds targeted to the ATP-site as well as the protein substrate site of PKAc), and by co-crystals of PKAc with representatives of ARC-III.
The profiling of representatives of ARC-II and ARC-III towards a panel of PKs indicated that the novel ARCs were group-selective inhibitors possessing high affinity towards several PKs of the AGC-group; the more detailed studies with PKAc, PKGI, PKB and ROCK-II revealed several structural elements (e.g., nucleosidic fragment or chiral moiety) which influence the inhibitory potency and enable tuning the selectivity of ARCs.
The wide applicability of novel ARCs was confirmed by development of ARC-based biochemical in vitro assays for PKGI with detection of change of fluorescence anisotropy (FA), fluorescence intensity (FI), or FRET. While the FA-assay was adapted from the previous PKAc studies, the FI- and FRET-assays were originally designed.
The potential of novel ARCs as physiological modulators was de-monstrated in the internalization studies with the isolated smooth musc-le tissue, and in vasoconstriction experiments with isolated rat arteries (via targeting cAMP/PKA and cGMP/PKG pathways).
SUMMARY IN ESTONIAN
Adenosiini analoogi ja oligoarginiini konjugaatidel põhinevad proteiinkinaaside inhibiitorid
Valkude fosforüleerimisreaktsioon elusorganismides ning seda katalüüsivad valgud – proteiinkinaasid – on olnud loodusteaduste tähelepanu keskpunktis alates 1950-test aastatest, kui E. P. Kennedy, E. H. Fischer’i, E. G. Krebs’i, E.
W. Sutherland’i ja W. D. Wosilait’i tööd avasid kinaaside ajastu [Cohen P., 2002]. Proteiinkinaasid osalevad enamikes raku elutegevuseks olulistes protses-sides, seetõttu on häired nende ensüümide funktsioneerimises (eriti kõrgendatud aktiivsus) seotud mitmete haigustega (sealhulgas vähkkasvajad, diabeedid ja astma). Sellest tulenevalt keskenduvad nii akadeemiliste teadlaste kui ka far-maatsiafirmade ravimiarendajate huvid üha enam kinaaside toimemehhanismide uurimisele ja võimaluste selgitamisele rakusiseste valkude fosforüleerimistasa-kaalude mõjutamiseks. Viimase ülesande lahendamisel on üheks perspektiiv-semaks teeks proteiinkinaaside inhibiitorite arendus.
Käesolev töö kirjeldab adenosiini analoogi ja arginiinirikka peptiidi konju-gaatide (ARC-de) arendamist proteiinkinaaside bisubstraatseteks kõrge inhibeerimispotentsiaaliga ligandideks, nende disaini ja sünteesi. Uudsete ARC-de arendamisel lähtuti tulemustest, mis saadi inhibiitorite struktuuri mõju uuri-misel nende sidumis- või inhibeerimisomadustele cAMP-sõltuva proteiinkinaa-siga (PKA) ning sama proteiinkinaasi ARC-dega kooskristalliseerimisel saadud struktuuride röntgenanalüüsil. Töö tulemusena õnnestus valmistada kaks uut ARC-tüüpi inhibiitorite põlvkonda (ARC-II ja ARC-III), mille esindajatel on võrreldes lähteühenditega (ARC-I) järgmised erinevused:
ARC-II puhul amideeriti konjugaadi C-terminaalne karboksüülrühm ning peptiidses fragmendis asendati L-arginiinid D-arginiinidega; lisaks asendati mõnede ühendite nukleosiidses fragmendis adenosiini derivaat Hidaka inhi-biitori H-9 jäägiga. ARC-II kõige efektiivsema esindaja (ARC-903) IC50 väärtus PKAc inhibeerimiskatsetes oli 5,3 nM.
ARC-III puhul lisati konjugaatide struktuuri kiraalne element (aminohape) ja teine linker. ARC-III kõige efektiivsem esindaja oli ARC-1028, mille IC50 väärtus väljatõrjumiskatsetes PKAc-ga oli alla 0,5 nM.
Järgmise sammuna määrati ARC-II ja ARC-III kõige efektiivsemate ühendite selektiivsusprofiilid; ühendid osutusid rühm-selektiivseteks inhibiitoriteks mit-mete basofiilsete proteiinkinaaside, eriti AGC-rühma esindajate suhtes. De-tailsemates uuringutes proteiinkinaasidega PKGI, PKB ja ROCK-II tehti kindlaks ka uute ARC-de struktuurifragmendid, mis mõjutavad inhibiitorite selektiivsust ühe või teise kinaasi suhtes.
Edasistes uuringutes tehti kindlaks, et tulenevalt uute ARC-de suhteliselt laiast selektiivsusprofiilist oli võimalik rakendada ARC-del põhinevat sidumis-meetodit (FA-meetod) kinaasiaktiivsuse hindamiseks ning proteiinkinaasi-inhibiitorite iseloomustamiseks mitte ainult eelnevalt mudelkinaasina kasutatud
PKA jaoks, vaid ka viimasega struktuurselt sarnase PKGI jaoks. Lisaks eelne-vale arendati uute ARC-de põhjal PKGI jaoks veel kaks sidumismeetodit, milles inhibiitorite afiinsuse või kinaasi aktiivsuse määramiseks mõõdeti fluo-restsentsi või FRET-i intensiivsuse muutust. Kõiki eespoolmainitud meetodeid on suure tõenäosusega võimalik kohandada ka mitme teise basofiilse proteiin-kinaasi jaoks.
Töö viimases etapis demonstreeriti, et uute ARC-de rakendusala ei piirdu biokeemiliste in vitro mõõtmismeetoditega, vaid see on laiendatav in vitro katsetele elusrakkudes ja -kudedes. ARC-II ja ARC-III efektiivsemate esin-dajate proteolüütiline stabiilsus ja võime läbida raku plasmamembraani võimal-dasid uurida uusi inhibiitoreid roti isoleeritud ajuarterite silelihaskoes. Nende katsetuste tulemusena selgus, et ARC-d suudavad inhibeerida cAMP/PKA ja cGMP/PKG radu füsioloogilistes tingimustes.
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